Method for the simultaneous dissection in specific positions of filiform organic molecular chains, in particular dna

ABSTRACT

The invention relates to a method for the simultaneous dissection in specific positions of filiform organic molecular chains, in particular DNA. The aim of the invention is to provided a method, by which a highly specific dissection can take place on certain sequences that can be freely selected and simultaneously on numerous filiform molecules. To achieve this, nanoparticles (1) are provided with a molecular chain (11) of any predeterminable sequence, which is selected to be complementary to a sequence of a molecule (2) that is to be dissected, said molecular chain(s) (11) is/are hybridised in the usual manner with the molecule, or specifically linked to said molecule in another manner and the nanoparticles (1) are subsequently subjected to a high-energy radiation of at least one wavelength, which can be absorbed by said nanoparticles (1).

BACKGROUND OF THE INVENTION

The invention relates to a method for the simultaneous dissection inspecific position of filiform organic molecular chains, in particularfor the sequence-specific dissection of DNA. The dissection of DNA atpredetermined positions is a fundamental technique of molecular biology.To date, in routine laboratory works only such enzymes are used(restriction enzymes) which have a specific dissection sequence. Theenzyme EcoRI, for example, dissects double-stranded DNA at all positionswith the following base sequence: 5′ GAATTC 3′ 5′ GAATTC 3′Such dissections are used in routine work for the manipulation of DNA,for example for the integration of new fragments or for a definedreduction. A complete field of DNA analytics, known as fingerprinting,is based on the variation of length distribution of DNA after the use ofsuch enzymes, the length distribution is documented in the restrictionfragment length polymorphism. If there is a mutation in the field of onedissection sequence in singular individuals (that is for example an Ainstead of a T in the scheme given above), the DNA cannot be dissectedat this position, and DNA fragments develop which have other lengthsthan the ones in individuals without mutation at this position.Therefore, the pattern of the length distribution can be used foridentifying the individual man. This fact is made use of for forensicpurposes or for paternity affiliations. The extremely high sensitivityof this technique applied for this purposes (the change of an individualbase can be detected) is also used for the determination of geneticdefects (such as hereditary diseases) which are localized on thedissection sequences. When using all these techniques, however, one isrestricted to the naturally existing enzymes and their dissectionsequences, other positions cannot be dissected in this manner.

To avoid such restrictions single molecule based techniques have beendeveloped for dissecting DNA at any positions by using a laser beam[Schütze, K., I. Becker, et al. (1997) “Cut out or poke in the key tothe world of single genes: laser micromanipulation as a valuable tool onthe look-out for the origin of disease” Genetic Analysis 14(1): 1-8)] orby using the atomic force microscope [AFM, Henderson, E. (1992) “Imagingand nanodissection of individual supercoiled plasmids by atomic forcemicroscopy” Nucleic Acids Research 20(3): 445-447]. These two methodshave the significant disadvantage that they do not offer selectivity andare characterized by a large dissection width. Laser cutting destroysseveral hundreds of base pairs and generally an orientation can only beachieved on the basis of typology (start/end) or by means of afluorescence-marked DNA fragment (FISH: fluorescence in situhybridization), whereby the optic resolution (>100 . . . 200 nm) limitsthis method. In addition to this, both methods are single molecule basedtechniques and do only allow to dissect a single molecule instead of anumber of molecules according to the lab standard. Thus, acharacterization (for example gel-electrophoresis) or a furtherprocessing requires a multiplication in order to be compatible with thestandard laboratory methods.

To avoid the limitation caused by the restricted spatial resolution ofsuch physical methods, the proximity focusing technique has been used inmaterial processing. This technique uses a small object (a scanning tipof an atomic focusing microscope having a radius in the lower nanometerrange) as a high-intensive secondary light source which is supplied byradiated laser light [Gorbunov, A. A. and W. Pompe (1994) “Thin FilmNanoprocessing by Laser/STM Combination” phys. stat. sol. (a) 145:333-338]. This secondary radiation becomes effective in the vicinity ofthe small object (near-field effect), and thus a focusing effect in thesize of the object is achieved.

SUMMARY OF THE INVENTION

The aim of the invention is to provide a method by means of which ahighly specific dissection can take place on certain sequences that canbe freely selected and simultaneously on numerous filiform molecules, inparticular DNA. This aim is achieved by a specific marking by means ofnanoparticles which are subsequently subject to energy radiation. Theinvention is based on the principle that energy is sent into the samplein a broader beam, but due to an appropriately placed nanoobject it hasonly an influence on a small volume area. This effect is achieved by thenanolocal conversion of the radiation energy that is absorbed by thenanoobject into heat and by chemical conversion.

In fact, single molecules such as dyes can be positioned well in thenanorange, but their efficiency for local absorption and energyconversion is low. Moreover, due to the absorption of the radiation theyoften lose their absorbing effect very rapidly in photochemicalprocesses. This invention, however, uses electron-conductingnanoparticles, metallic nanoparticles in particular, and here the onesfrom heavy precious metals especially. Unlike dye markers they can beused in a broad spectral range, too and are not limited to a small one.Apart from electromagnetic radiation (IR radiation, visible light, UVlight, X-radiation), the energy of the radiation of high-speed particlessuch as electrons or ions can be effectively converted, too. In thefield of optics, the efficiency of the radiation conversion is increasedthanks to the narrow intensive bands of the plasma resonance. By workingwith particles which consist of the same material but have differentdimensions, plasma resonance can be used for various wave lengths toaddress local cutting positions additionally or to obtain locally verylimited dissections by the cooperative effect of two or more wavelengths.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail by way of thefollowing schematic example. The figures show:

FIG. 1 a-e the schematic flow of the process by using one singlemolecular strand,

FIG. 2 a-c demonstrates certain difficulties when dissectinglong-stretched molecules which show a three-dimensional folding and

FIG. 3 a-c shows the possible solution for avoiding the difficultiesaccording to FIG. 2 a-c.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the present invention nanoparticles in the range of between 1 nm and150 nm are used. In the example, gold nanoparticles 1 having a typicaldiameter of 15 nm are placed along a DNA strand 2 by a specific linkageto the sequence desired and are then subject to the radiation of theappropriate kind of energy (light). For this purpose, the nanoparticles1 are first provided with a short DNA molecule 11 with more than tenbases, whereby only four of them are shown in FIG. 1 b and c to allow aclearer overview. The sequence of this molecule is complementary to theDNA 2 to be dissected (target DNA, see FIG. 1 a, for which a partsequence of a single strand is given as example in enlargedpresentation) at the dissection point. In this way, a specific link ofthe nanoparticle 1 at a defined place along the DNA 2 is achieved (seeFIG. 1 c, whereby the specific linkage is again shown in enlargedpresentation). Afterwards, one, preferably more DNA-moleculesspecifically marked in such a way is/are subject to high-energyradiation, for example to the light of an argon laser at one or morewave lengths, to address different nanoparticles which are linked tovarious sequence fragments. Due to the energy absorption at thenanoparticle, the particle and the environment are heated up locally inthe way demonstrated schematically in FIG. 1 d. This heat causes thedissection of the DNA strand. (See FIG. 1 e.) The precision of thedissection (=width of dissection) can be controlled finely by adjustingthe radiated energy. A target precision (=location of dissection) in thelower nanorange is ensured by the self-organizing system of particleswith any predeterminable DNA linking sequence, which link to thedissection area(s) on the target DNA by hybridization (copulation ofcomplementary DNA fragments). This hybridization can take place forexample after the local opening (melt open) of the DNA double-strand inform of a double-strand or by the addition of the linkage sequence tothe double-strand by forming a triplex-structure (energetically favoredunder certain conditions depending on salt content and base frequency inparticular). This invention has the considerable advantage that theenergy can be radiated onto a larger area. From the technological pointof view, it is therefore less complicated than the conventional methods.

For the long-stretched DNA molecules a problem is caused by theirtypically three-dimensional folding. As shown in FIG. 2 a-c, someadditional dissections can be produced unintentionally despite a precisepositioning, because other parts of the molecule 2 come close to thenanoparticle excited 1 due to the back-folding of the strand. A certaincontrol of the back-folding is possible via the salt concentration ofthe solution, because it influences the rigidity of DNA. A globularstructure can be avoided by stretching the molecule 1, but it requiressingle molecule based techniques (optical tweezers) or othertime-intensive methods such as liquid stream and/or electrical fieldpreferentially combined with a unilateral binding.

A simple and highly parallelizable solution variant is given by thelinkage of the nanoparticles 1 on suitable surfaces 4. For this purpose,the substrate material used has to offer such a quality that theabsorption of the radiated energy is not considerable, for example glassif light is used to excite the nanoparticles. For the present inventionthe nanoparticles 1 are formed by spots structured on the surface 4.After the immobilization of the nanoparticles 1 with short DNA 11 on thesurface 4, whereby, for example, in the case of using glass its surfacecan be coated with silane or provided with another coating allowing theadhesion of the particles, the DNA 2 settles according to the scheme ofthe specific linkage by DNA base coupling (hybridization) described inFIG. 1. Now, the DNA molecule 2 cannot surround the nanoparticle 1 inspace any longer, because the back-folding chances are limitedconsiderably. Thus, the probability of unintentional multi-dissectionsshown in FIG. 2 is minimized.

Advantageous applications of the recommended method are the followingones:

-   -   the restriction analysis of DNA (such as restriction fragment        length polymorphism). By means of the procedure recommended        these analyses can be performed for special genes in an        optimally adjusted manner. A lot of genes being outside the        known enzyme dissection areas can now be reached.    -   the deliberate cutting of gene fragments out of bigger DNA        molecules. For this purpose, the cutting sequences can be        selected in such a way that the searched fragment can be simply        isolated (for example by a unique length) and has not to be        separated from a number of DNA fragments which have the same or        a comparable length.    -   the controlled release of DNA fragments. For this purpose, a        procedure is known according to the prior state of technology in        which surface areas with double-stranded DNA are heated up by        laser energy to such an extent that the double-strand will be        opened (melt open). In this method, temperatures above the        melting point (50-95° C.) are to be generated locally without        reaching temperature peaks which damage the molecules. Based on        the invention recommended this complicated temperature regime        can be replaced by the use of immobilized colloids which link        the molecules with the dissection sequence. (See FIG. 3 a.)        These molecules are dissected by a special exposure (FIG. 3 b)        and the fragments are released (FIG. 3 c). Moreover, this        procedure can be parallelized by linking several dissection        sequences in various surface areas which are than subject to        light independently from each other. In this way, the        appropriate fragments are released as defined.

The invention is not limited to the sequence specific dissection of DNAdescribed in the examples, but it can also be used for other biopolymerswhich allow a position-specific linkage of the nanoparticles providedwith suited linking partners. Moreover, the kind of radiation used candiffer from the one described here, as far as the nanoparticles usedabsorb the energy radiated.

The nanoparticles can be formed by metal and semiconductor particles orby composites of these materials and they can include organiccomponents. The diameter of the particles can be adjusted by aseparation procedure, for example by separating metal, semiconductor ororganic materials.

The use of different dimensional classes of particles in combinationwith appropriately adjusted sources of radiation allows the independentprocessing of various subsets, as described in FIG. 4 b for DNA. Byusing different particle classes, two in this example, which differ fromeach other in their energy absorption (i.e., caused by their differentdiameters), it is possible to activate only one class deliberately byselective radiation.

1. Method for dissection in specific positions of a plurality offiliform organic molecular chains, comprising providing respectivenanoparticles with molecular chains complementary to a sequence of thefiliform organic molecular chains that are to be dissected, linking thecomplementary molecular chains complementarily to respective saidfiliform organic molecular chains and subsequently subjecting thenanoparticles to high-energy radiation of at least one wavelength whichis absorbed by said nanoparticles.
 2. Method according to claim 1,wherein the respective nanoparticles are of at least two differenthigh-energy radiation absorption characteristics and the respectivedifferent nanoparticles are provided with respective different molecularchains complementary to respective different sequences of the filiformorganic molecular chains, each respective different molecular chain withwhich a respective different nanoparticle is provided being identical.3. Method according to claim 2, wherein the respective differentnanoparticles are of identical composition but of respective differentsize.
 4. Method according to claim 1, wherein the nanoparticles aremetallic.
 5. Method according to claim 1, wherein the high-energyradiation comprises diffusely radiated high-energy light.
 6. Methodaccording to claim 1, wherein the respective nanoparticles are of atleast two different compositions and the sequences of the complementarymolecular chains with which all the nanoparticles are provided areidentical.
 7. Method according to claim 1, wherein the nanoparticlesprovided with complementary molecular chains are immobilizedunilaterally on a surface.
 8. Method according to claim 7, wherein thesurface comprises a material which absorbs substantially less of saidhigh-energy radiation than is absorbed by said nanoparticles or none ofsaid high-energy radiation.
 9. Method according claim 7 or 8, whereinthe nanoparticles comprise spots on the surface.